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Neolignans from Nectandra megapotamica (Lauraceae) Display in vitro Cytotoxic Activity and Induce Apoptosis in Leukemia Cells.
Ponci V
,
Figueiredo CR
,
Massaoka MH
,
de Farias CF
,
Matsuo AL
,
Sartorelli P
,
Lago JH
.
Abstract
Nectandra megapotamica (Spreng.) Mez. (Lauraceae) is a well-known Brazilian medicinal plant that has been used in folk medicine to treat several diseases. In continuation of our ongoing efforts to discover new bioactive natural products from the Brazilian flora, this study describes the identification of cytotoxic compounds from the MeOH extract of N. megapotamica (Lauraceae) leaves using bioactivity-guided fractionation. This approach resulted in the isolation and characterization of eight tetrahydrofuran neolignans: calopeptin (1), machilin-G (2), machilin-I (3), aristolignin (4), nectandrin A (5), veraguensin (6), ganschisandrin (7), and galgravin (8). Different assays were conducted to evaluate their cytotoxic activities and to determine the possible mechanism(s) related to the activity displayed against human leukemia cells. The most active compounds 4, 5 and 8 gave IC50 values of 14.2 ± 0.7, 16.9 ± 0.8 and 16.5 ± 0.8 µg/mL, respectively, against human leukemia (HL-60) tumor cells. Moreover, these compounds induced specific apoptotic hallmarks, such as plasma membrane bleb formation, nuclear DNA condensation, specific chromatin fragmentation, phosphatidyl-serine exposure on the external leaflet of the plasma membrane, cleavage of PARP as well as mitochondrial damage, which as a whole could be related to the intrinsic apoptotic pathway.
Figure 1. Tetrahydrofuran neolignans 1–8 isolated from the leaves of N. megapotamica.
Figure 2. (A) Morphological changes induced by 4, 5 and 8. HL-60 cells were incubated with 50 µg/mL of each compound for 24 h and analyzed by light microscopy. The formation of blebs in cell membrane was evidenced in all HL-60 treated cells. Magnification ×400; (B) Chromatin condensation and fragmentation analysis by fluorescence microscopy. 1 × 105 HL-60 cells were incubated with 50 µg/mL of 4, 5 and 8 for 24 h and stained with DAPI (blue) for chromatin analysis (original magnification, ×400). White arrows indicate chromatin condensation and fragmentation process; (C) DNA analysis of HL-60 cells after incubation with 50 µg/mL of 4, 5 and 8 for 24 h. DNA was extracted from tumor cells and analyzed in 1% agarose gel; (D) Translocation of phosphatidylserine in B16F10-Nex2 (5 × 105 cells) previously incubated with compounds 4, 5 and 8 at 100 µg/mL and negative control (RPMI medium) for 24 h. AV−PI− (live cells); AV+PI− (early apoptotic cells); AV−PI+ (necrotic cells); AV+PI+ (late apoptotic cells).
Figure 3. (A) Mitochondrial membrane depolarization induced by compounds 4, 5 and 8 in HL-60 cells; (B) Lysates from HL-60 cells, previously treated with compounds 4, 5 and 8 at 100 µg/mL for 24 h at 37 °C, were analyzed by Western blotting. Antibodies against cleavage PARP and β-actin (protein loading control) were used.
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